All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Microbial detection and instigation of an appropriate innate and subsequent adaptive immune response to a pathogenic assault, is highly reliant on toll-like receptors (TLRs) (Brikos et al. (2008) HANDB EXP PHARMACOL, 21-50). Members of the TLR family recognize specific conserved pathogen-associated molecular patterns (PAMPs) expressed by invading microorganisms. TLR4, one of the most widely studied TLRs, recognizes a repertoire of PAMPs, which includes lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria (Poltorak et al. (1998) SCIENCE 282, 2085-2088; Qureshi et al. (1999) J EXP MED 189, 615-625; Hoshino et al. (1999) J IMMUNOL 162, 3749-3752; Medzhitov et al. (1997) NATURE 388, 394-397). For optimal LPS-induced signal transduction to occur, a receptor complex is assembled consisting of the signaling subunit TLR4, the co-receptor myeloid differentiation (MD)-2, and two accessory proteins, LPS-binding protein (LBP) and CD14 (Schumann et al. (1990) SCIENCE249, 1429-1431; Pugin et al. (1993) PROC NATL ACAD SCI USA 90, 2744-2748; Frey et al. (1992) J EXP MED 176, 1665-1671; Shimazu et al. (1999) J EXP MED 189, 1777-1782).
MD-2 belongs to the MD-2-related lipid recognition (ML) family (Inohara et al. (2002) TRENDS BIOCHEM SCI 27, 219-221). A secretion signal, the signature sequence of this group of proteins, is located at the N-terminal domain of MD-2 (Kato et al. (2000) BLOOD 96, 362-364). Although MD-2 lacks transmembrane and intracellular regions, it may be membrane-bound through its association with the extracellular portion of TLR4 (Akashi et al. (2000) J IMMUNOL 164, 3471-3475). Studies with mice deficient in either MD-2 or that lacked a functional TLR4 have revealed that both proteins are absolutely required for LPS signaling (Poltorak et al. (1998) SCIENCE 282, 2085-2088; Qureshi et al. (1999) J EXP MED 189, 615-625; Hoshino et al. (1999) J IMMUNOL 162, 3749-3752; Shimazu et al. (1999) J EXP MED 189, 1777-1782). Although TLR4 is critical to mount a response to gram-negative bacteria, tight regulation of the TLR4 signal transduction pathway is imperative to prevent excessive inflammation that could lead to collateral damage to the host (Liew et al. (2005) NAT REV IMMUNOL 5, 446-458). One method of control involves alternative splicing of specific genes that encode essential components of the TLR4 signaling pathway to produce inhibitory isoforms, examples include myeloid differentiation factor 88S (MyD88S) (Janssens et al. (2002) CURR BIOL 12, 467-471), and smTLR4 (Iwami et al. (2000) J IMMUNOL 165, 6682-6686; Jaresova et al. (2007) MICROBES INFECT 9, 1359-1367). Similarly, the murine MD-2 gene encodes two alternatively spliced isoforms. The truncated variant, MD-2B, generated by the splicing out of the first 54 amino acids of exon 3, downregulates LPS signaling (Ohta et al. (2004) BIOCHEM BIOPHYS RES COMMUN 323, 1103-1108). Given that mouse and human MD-2 are highly conserved, an alternative splicing of this gene in humans could also play an important regulatory role in humans.
There is still a need for therapeutic strategies that may be used to treat human pathologies characterized by an overly exuberant or chronic immune response to LPS. Therefore, novel mechanisms to further regulate TLR4 signaling would be beneficial and the protein MD-2s described herein may be used to treat these human pathologies.